Botulinum neurotoxins (BoNTs) are among the most poisonous substances known to man, with a 50% lethal dose (LD50) of 1 ng/kg in mice. BoNTs therefore represent a major bioterrorist threat (Arnon et al., 2001; Gill, 1982). Paradoxically, BoNT-containing medicines and cosmetics, such as Botox(R), Dysport(R), Xeomin(R), CBTXA(R), Myobloc(R) and NeuroBloc(R), have been used with great success to treat a variety of neurological, otolaryngological, ophthalmological, urological, dermatological and gastrointestinal disorders (Jankovic, 2004; Truong and Jost, 2006). In October 2010, FDA approved Botox(R) to treat chronic migraine. Both the toxic and therapeutic functions of BoNTs rely on a common mechanism to enter neurons, cleave proteins that mediate exocytosis of key neurotransmitters, and subsequently paralyze the affected muscles. BoNTs are produced by Clostridium botulinum as large progenitor toxin complexes (PTCs) that include auxiliary clostridial proteins known as neurotoxin-associated proteins (NAPs) (Montecucco and Schiavo, 1995). Clinical formulations are also composed of BoNT PTCs. Accidental BoNT poisoning mainly occurs through oral ingestion of tainted food products. NAPs are thought to protect BoNTs against the hostile environment of the gastrointestinal (GI) tract, and to mediate toxin transport across the epithelial cell barriers, the first step of intoxication. However the underlying molecular mechanisms that control these processes are poorly understood. The goal of this proposal is to elucidate the structure and function of BoNT PTCs, including the mechanism by which NAPs protect BoNTs, the regulatory mechanism underlying PTC assembly, and the structural basis by which NAPs regulate the interaction between BoNTs and host cells. The focus of this proposal is on the serotype A of BoNT (BoNT/A), because it is a major concern for bioterrorism and is the most commonly used medicine among all BoNT serotypes. We will use a combination of techniques, including X-ray crystallography, together with biochemistry, biophysics, cell biology, toxicology and bioinformatics. If successful, the proposed wok will guide the design of novel therapeutics for the treatment and/or prevention of botulism. Our work may also provide new insights into the activity and side effects of drugs such as Botox(R) and Dysport(R), which may help improve their clinical efficacy and suggest novel clinical applications.